A biological specimen (sample) chosen for microscopic analysis, can be, for example, a tissue suspected of having a particular disease. An example of a biological sample is a tissue biopsy of a suspected cancer lesion. The biopsy may then be studied to see if it contains a particular molecular marker, or target of interest. If the target of interest is present in the biopsy then the diagnosis of cancer is confirmed, whereas if the target of interest is not present in the biopsy then the diagnosis of cancer may be ruled out.
A conventional method of preparing a biological specimen for microscopic analysis can comprise the following steps. A tissue sample is obtained and placed into a fixative for a period of time sufficient to fix it, rendering it rigid. The fixed tissue sample is dehydrated to remove all water and then embedded into a suitable embedding medium, such as paraffin. Thin sections of the tissue sample are cut from the paraffin block. Each tissue sample section is cut sufficiently thin (for example, about 4-6 u) so that it can be examined by a microscope. The thin sections are laid onto a microscope slide where they adhere to the slide via charged interactions. The paraffin in the tissue sample is then removed through the use of paraffin solvents, such as xylene, leaving the tissue behind. The tissue sample is then rehydrated by passing through a series of graded alcohols and water. Once the tissue sample (specimen) is rehydrated, it is ready for staining. Typically staining is achieved by exposing the tissue sample to one or more series of reagents (dyes/stains/chromogens). The end result of the staining is that colored areas of the tissue sample are produced. These colored areas provide a trained microscopist, such as a pathologist, with information that can be used to render a diagnosis. Frequently these stains are accurate enough to selectively stain specific molecular targets within the tissue sample, or within individual cells. Stains with this level of specificity include immunohistochemistry, immunocytochemistry, and in situ hybridization. Prior to mounting the stained biological sample on the slide, the water contained in the stained biological sample must be removed by a process called dehydration. After dehydration, a mounting medium is placed on the stained biological specimen. The mounting medium typically contains an organic solvent. Mounting the stained biological specimen improves its optical resolution and renders it acceptable for microscopic analysis. Lastly, a glass coverslip is placed onto mounting medium.
Several problems currently exist regarding the conventional methods of preparing a biological specimen (sample) for microscopic analysis.
The first problem exists in the dehydration step that occurs just before mounting the slide. The dehydrating fluid typically used is an alcohol, such as ethanol, which is soluble in water and can penetrate into the stained biological specimen and replace the water within the specimen with alcohol. Then the alcohol is replaced with an alcohol-miscible organic solvent, such as xylene, which can then penetrate into the tissue of the specimen. The alcohol-miscible organic solvent prepares the specimen for accepting a mounting medium that also contains an organic solvent. These steps are necessary because a stained biological specimen, which contains water, cannot be directly mounted into an organic mounting media (resinous mounting media) without first removing the water from the specimen. Because of the toxicity and health concerns associated with the use of alcohols and other organic solvents it is preferable to use other methods of dehydration, such as air-drying. However, air-drying frequently produces suboptimal drying artifacts, such as nuclear distortion and shrinkage.
Second, biological specimens (samples) that have been stained in preparation for microscopic analysis may be saved for re-evaluation over a period of months or years. Frequently during this extended period of storage the stains exhibit fading. Fading can be caused by prolonged exposure of the stains to the overlaying mounting medium and/or exposure to light.
Third, only stains that are resistant to organic solvents (e.g. alcohol-miscible organic solvents) used during the dehydration step, and organic solvents used during the mounting step can be utilized due to the dissolving action of organic solvents on stains. This greatly limits both the number of stains and mounting media that can be used.
What is needed is a sealing fluid that does not require dehydration or drying of a stained biological specimen prior to mounting. What is also needed is the use of a sealing fluid that decreases the use of alcohols or organic solvents, thus reducing the risk of exposure to the user. Lastly, what is needed is a sealing fluid that can be used with any stain and any mounting media.
Disclosed herein are novel sealing fluids (compositions) for sealing a biological specimen on a slide for microscopic analysis. The disclosed sealing fluids allow a biological specimen to be dehydrated without the use of organic solvents, protects the biological specimen from drying artifacts during a drying process, and can be used with any stain or any mounting media due to a protective barrier formed by the sealing fluid between the mounting medium and the stained biological specimen. The disclosed sealing fluids protect stains performed on biological specimens from becoming dissolved by organic solvents present in the mounting media, and protects stains performed on biological specimens from fading over time.